Many acid-base transporters contribute to inll'acellular and extracellular pH regul ation in brain. Electroge nic Na/Bicarbonatc Cotransp0l1crs (NBes) arc particu larly imp0l1ant because they al ter pH in response to neuronal activity. While the physiologic significance of multiple NBC vnriants in brain and other tissues is not known, many of these variants exhibit different regulatory profiles. NBCs are regulated by classic second messengers involving protein kinases A and C, as well as interacting proteins. However, the role of other regulatory pathways involving phospholipids for instance has 110t been elucida ted. '111e longterm objective of this proposal is to identify and characterize the regulatory mechanisms of Na+-coupled bit:arbonatt! transporters (BTs) in an effort to understand the significance of multiple 13Ts nnd the Illo leculnl'u'lsis of tramipo rler function. Patch-clamp techniques and fluo rcsccnce imaging will he used to exam inc phosphatidylinositol 4,S- hisphosphnte (PIP:!) regulation of pHi and ncid extrudcrs (e.g., NBCs, the Nadrivcn CI-HCO,j exchanger (NDCD E), and )./a-H exchangers (NHEs)) in cult ured astroc)'tes and neurons from rat hippocampus (Aim I). Int racellular P I P ~ levels will be altered in cells by di rect perfusion. b~lth incubation with cell ~ p e r m cant PI P", and transfection with phosphoinositide enzymes. Changes in cell ular PIP" will be <lsst!ssed by mass spectroscopy and a protein~ l ipid overlay assay. Characteristics to be examined include PJ P~ ECso, Na~ and voltuge dependencies, and sensitivity to charge scrccners and PIP2 blocJ.;ing nn tibodies. According to preliminary data, PIP" stimulates the activity of NBCe l va riants by modulating the rcgulatOlY role of thei r cytoplasmic amino termini (Aim 2). In Aim 2 , two-eleetrode voltage-damp and macropatch techniques with Xenopus ooC)"tes heterologously expressing trunca ted "mel mutant i\BCt'l va riants will be uscd to identify and characterize N terminal regions/ residues th at modulate transporter fUllction and PIP:: sensiti\ity. PIP ~ dose-response eurves will be generated. The effect of PIP:! on the biophysical properties (e .g., KM val lies for tl'ansported ions, Vlll~.~ va lu~ s, and current-voltage relationships) of NBCCl variants will be assessed. The effect of peptides containing id enti fied rcglliatory regions on NBC fU ll ction will be 'lssessed. The results from these aims will elucidate PIP~ as a novel regulatory mechanism of NBC activi ty that likely provi des a permissi\'c pH environment for PIP2 targets that modulate neuronal t'xcitahility. Characterizing the molecular basis of P[P2's sti mulation of NBCel extends our lmderstanding of tht! importance of multiple NBCs, and provides mechanistic and st ructural insight in to ~mc function. The information \,;;1\ enhance our understa nding of acid -base handling by brain cells, partieul arly in acid-base disturbances associ:Jted \,;;th ischemia, anoxia/ hypoxia, stroh, and reperftlsion inj ury.